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Remodeling of Vaginal Connective Tissue in Patients With Prolapse

Author Information

From the Magee-Womens Research Institute and the Department of Obstetrics & Gynecology at Magee Womens Hospital and Center for Biological Imaging at the University of Pittsburgh, Pittsburgh, Pennsylvania.

Supported by a grant (R01 HD NIH 045590) from the National Institutes of Health The authors thank Mary Ann Portman, Michael Lupenetti, Carol Krupski, and Sonya Noh for contributing to tissue procurement from the control population.

Abstract

OBJECTIVE: As pelvic organ prolapse progresses, the morphology of the vagina dramatically changes. The objective of this study was to determine whether these changes observed clinically correlate with histologic and biochemical evidence of tissue remodeling

METHODS: After informed consent, full-thickness biopsies of the vaginal apex were obtained at the time of surgery from 77 women. The tissue of 15 premenopausal women with less than stage II prolapse (controls) was compared with that of 62 women with prolapse divided according to their menopausal status. All specimens were examined histologically. Scanning confocal microscopic analysis of fluorescent micrographs was used to quantitate collagen subtypes I, III, and V. Collagen fiber orientation was analyzed by scanning electron microscopy. Gelatin zymography was used to quantitate the expression of the proenzyme and active forms of matrix metalloproteinases (MMP) –2 and –9. Median values were compared using Mann-Whitney U or Kruskal-Wallis tests, where appropriate

RESULTS: Vaginal collagen fibers are arranged in a whorled pattern, with collagen III as the predominant fibrillar collagen. The amount of total collagen in the vagina was increased in women with prolapse relative to women without prolapse (P = .054) primarily due to increased expression of collagen III (P = .031). There was no difference in the expression of proMMP-2, active MMP-2, or proMMP-9; however, active MMP-9 was increased in patients with prolapse (P = .030)

CONCLUSION: The increase in collagen III and active MMP-9 expression in the vaginal tissues of patients with prolapse suggests that this tissue is actively remodeling under the biomechanical stresses associated with prolapse.

Level of Evidence: II-2

Prolapse of the pelvic organs is a common disease affecting the lives of millions of women. Women with prolapse suffer from chronic pelvic pain and pressure, urinary and fecal incontinence, sexual dysfunction, and social isolation.1,2 The lifetime risk of undergoing a surgery to correct pelvic organ prolapse or urinary incontinence is 11%.3 Surgical failures are relatively common, with 30% of women who undergo a primary surgical repair requiring additional surgery.3 Roughly 200,000 women have surgery to repair prolapse in the United States annually,4 with a cost to society for repair of prolapse that is estimated to exceed 1 billion dollars per year.5 Despite the high prevalence of this disease and the devastating impact on the lives of women, very little is known its pathophysiology.

In addition to the levator ani muscles,6 the vagina and its supportive tissues provide one of the primary mechanisms of support to the pelvic organs (bladder, uterus, and rectum). The vagina itself is composed of 4 layers: a stratified squamous epithelium, a subepithelium (also referred to as the lamina propria), a layer of smooth muscle referred to as the muscularis, and an adventitia. The vaginal subepithelium and muscularis form a fibromuscular layer beneath the vaginal epithelium, providing longitudinal and central support.7 Portions of the subepithelium, muscularis, and adventitia are plicated in gynecologic surgery to repair prolapse of the bladder or rectum into the vaginal canal.

For decades, it has been speculated that a structural defect in the vagina and its supportive tissues, such as a decrease in collagen content or a change in collagen subtypes, is one of the mechanisms that predisposes a woman to prolapse. Thus, there are multiple studies throughout the literature in which collagen is analyzed in vaginal biopsies procured at the time of a repair of prolapse in patients with prolapse or at the time of a hysterectomy in patients without prolapse is compared, with conflicting results.8–15 In most studies to date, the specimens have not been clearly defined histologically. As a result, it is uncertain which layers of the vagina are actually being analyzed, which in turn makes it difficult to compare studies. Indeed, expression of a structural protein such as collagen may be very different in the epithelium and the adventitia. A second limitation of these studies is that when comparing a tissue that is prolapsed to one that is not prolapsed, it is impossible to distinguish between causes and effects of prolapse.

In the study outlined here, we use histologically defined specimens to provide evidence that the vaginal wall undergoes pronounced remodeling in patients with prolapse relative to patients without prolapse. To do this, we compare the expression of 2 groups of proteins that are the hallmark of tissue remodeling—fibrillar collagens and matrix metalloproteinases. The goal of the study is not to determine the cause of prolapse per se but instead to describe changes in the vaginal wall that are present in patients with prolapse. We believe, independent of cause or effect, information on changes that occur in the prolapsed vagina will be useful to surgeons who reconstruct the vagina in patients with prolapse, particularly when using native tissues. We focus this study on the vaginal subepithelium, muscularis, and adventitia because the epithelium plays minimal role in vaginal support and may contribute to misleading results. We hypothesize that the accelerated remodeling in patients with prolapse is due to the biomechanical stresses of the prolapsing pelvic organs on the vaginal wall.

MATERIALS AND METHODS

A total of 77 full-thickness vaginal wall biopsies were procured from women in the University Gynecology and gynecology private practices at Magee Womens Hospital undergoing a benign gynecologic procedure between March of 2001 and December of 2003. Written informed consent was obtained according to protocols approved by the Institutional Review Board at University of Pittsburgh. Eligible women were divided into 4 groups according to specific criteria.

Premenopausal women without prolapse or incontinence were used as the control group because it is uncommon for a postmenopausal woman to undergo a hysterectomy for a benign indication other than prolapse.16 We rationalized that the well-estrogenized premenopausal tissues would represent the “gold standard” for normal pelvic support. Thus, for inclusion in the control group (n = 15), women were required to be aged older than 24 years and younger than 55 years without prolapse (defined as < stage II by the Pelvic Organ Prolapse Quantification examination or Baden Walker classification system17,18) or incontinence, with regular menstrual periods over the preceding 12 months. Women in the premenopausal prolapse group (n = 16) were aged older than 24 years and younger than 55 years with stage II or greater prolapse and regular periods over the preceding 12 months. Premenopausal women taking oral contraceptives were included in the premenopausal group (n = 3; 2 controls and 1 premenopausal woman with prolapse). Postmenopausal status was defined as no menses over the previous 12 months. In the postmenopausal no hormone therapy prolapse group (n = 23), women were aged 50 years or older with stage II or greater prolapse and not on hormone therapy for more than 1 year. Women in the postmenopausal on hormone therapy prolapse group (n = 23) had stage II or greater prolapse and were currently on hormone therapy for more than 1 year. Women with incontinence (stress, urge, or mixed type), in addition to prolapse, were included in the prolapse groups. Exclusion criteria included the use of a progestin-only hormone regimen, history of pelvic malignancy or connective tissue disease affecting collagen or elastin remodeling, blood loss during surgery exceeding 500 mL, adhesions or scarring at the biopsy site, surgeon's judgement that a biopsy may harm the patient, history or presence of endometriosis, morbid obesity (body mass index [BMI] > 35 kg/m2), and inability to provide informed consent. The exclusion criteria were formulated for patient safety and to minimize factors that may influence endogenous hormone levels, collagen, or matrix metalloproteinase (MMP) expression. Control patients were required to have answered, “no” to the following questions, “Do you leak urine on a regular basis?” and “Do you experience bulging or something falling out you can see or feel in the vaginal area?” All women with prolapse, who answered “yes” to leakage of urine on a regular basis, completed the Medial and Epidemiological Aspects of Aging Questionnaire.19 Patients who answered “sometimes” or “often” to any of the questions were considered to have urinary incontinence. Subject demographic variables were collected by certified staff onto a data collection sheet and entered into a study database. Variables included age, race, ethnicity, BMI, gravidity and parity, maximal stage of prolapse, urinary incontinence, type of menopause (surgical or natural), date of last menstrual period, stage of menstrual cycle, previous hysterectomy, previous surgery for incontinence or prolapse, type of hormone therapy (estrogen or estrogen and progesterone), and smoking status (never, current, former). A Pelvic Organ Prolapse Quantification examination was performed on all patients with prolapse to define stage of prolapse.17 It is worth noting that the majority of control patients were under the care of physicians who were more familiar with the Baden Walker half-way system.16 Therefore, patients were categorized as control if their prolapse was less than half-way to the hymenal ring at maximal strain or less than stage II by Pelvic Organ Prolapse Quantification.

For biopsies, surgeons were asked to obtain a biopsy of full-thickness vagina (a minimum of 0.5 × 2.0 cm2 with no maximum) at one of the lateral fornices at the vaginal apex. The minimum size requirement was chosen based our prior experience of the amount of tissue that would be needed for biochemical assays. Of the 77 women biopsied, the biopsies were large enough (> 0.5 × 3.0 cm2) in 19 women to perform an analysis of both collagen subtypes and matrix metalloproteinase expression (6 controls, 5 premenopausal women with prolapse, 6 postmenopausal women with prolapse not on hormone therapy, and 2 postmenopausal women with prolapse on hormone therapy). The remaining smaller biopsies (≤ 0.5 × 2 cm2) were randomly assigned to undergo either collagen or MMP analysis (n = 58). In women undergoing a hysterectomy, full-thickness vaginal biopsies were obtained after the removal of the uterus. In women who did not have a uterus, a full-thickness biopsy of the vagina was taken after a colpotomy had been made. Biopsies were immediately passed off the surgical field to a trained technician in the operating room. For analysis by microscopy, full-thickness sections were imbedded and frozen in liquid nitrogen. For biochemical analysis, the epithelium and peritoneum were removed under a dissecting microscope and a portion of the dissected specimen was embedded for histologic analysis. The remaining dissected tissue was frozen in liquid nitrogen and stored at –80°C until use. Serial sections (5–7 μm) of full-thickness vagina were prepared for scanning electron microscopy or immunofluorescence as described below. Fiber orientation was determined using scanning electron microscopy. Quantitative analysis of total collagen and the relative amounts of collagen subtypes (I, III, and V) were performed using scanning confocal microscopic analysis of fluorescent micrographs. For biochemistry, the expression of proenzyme and active forms of MMP-2 and MMP-9 in the subepithelium, muscularis and adventitia were measured using substrate gelatin zymography.

For scanning electron microscopy, frozen sections of full-thickness vagina from 5 patients in each group were placed onto cover slips. The specimens were fixed in 2% glutaraldehyde in phosphate buffered saline for 1 hour, washed 3 times in phosphate buffered saline, then dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 95%, and 99.5%), followed by acetone. They were dried in a critical-point dryer with CO2. The specimens were mounted, coated with gold, and examined using a JEOL 6330F scanning electron microscope (JEOL, Peabody, MA).

For immunofluorescence, frozen embedded full-thickness vagina was cut into serial sections of 5–7 μm, placed onto gel-coated slides, and stored at –20°C until ready for use. Slides were fixed with cold acetone and blocked with Normal Donkey Serum for 45 minutes. The sections were then incubated simultaneously with 3 different primary antibodies (collagen I, III, and V). Optimal dilutions of antibody were determined by a series of previous titration experiments. Sections were then incubated with 3 secondary antibodies labeled with 3 distinct fluorophores and stained with Hoescht stain. To-Pro nucleic acid dye (Molecular Probes, Eugene, OR) was used to identify cell nuclei. Sections were then mounted with gelvatol and a cover slip and dried overnight at 4°C in the dark.

Scanning confocal microscopy was used to perform quantitative analysis of the 3 different fluorophores linked to collagens I, III, and V. By this technique, the focal volume collected in each image is constrained by the pinhole size, the numerical aperture of the objective, and the wavelength of the emitted light. Although the pinhole size can be altered, the remaining parameters are constants. In our analysis, the pinhole size is recorded by the software driving the microscope and is set at a predetermined optimal constant. With the imaging parameters maintained as a constant, the confocal becomes a highly quantitative fluorescence imaging tool.

Two individuals blinded to the study objectives analyzed the fluorophores in the subepithelium and muscularis of each sample. Scanning was performed using an Olympus Flowview BX61 confocal scanning laser microscope interfaced to a quantitative computer program (Metamorph 5.0, Universal Imaging Corporation, Downington, PA). A 40X/1.25 NA objective was used with 2x zoom and analysis was done based on the fluorescence of each fluorophore under the appropriate wavelength. Five to 7 sites were randomly selected within the subepithelium and muscularis of each sample. Fluorescence signals were digitalized to form a pixel-based image displayed on a monitor and recorded. Results are represented as mean voxel intensity per positive area as a proportion of the total sample area.

Quantitative gelatin zymography was carried out as described by Kleiner and Stetler-Stevenson.20 Briefly, dissected specimens that included vaginal subepithelium, muscularis, and adventitia were homogenized in a buffer containing 50 mM Tris HCL, pH 7.5, 0.2% sodium dodecyl sulfate, 1% Triton X-100, and protease inhibitors. Cell debris was removed by centrifugation at 12,000 g, and protein concentrations were determined in triplicate using a commercial kit (Biorad, Hercules, CA). Fifteen micrograms of protein was loaded on 10% acrylamide gels containing 0.15% gelatin under nonreducing conditions. After dialysis against a 2.5% Triton X solution, zymograms were incubated for 18 hours at 37°C in a buffer containing 200 mM NaCl, 5 mM CaCl2, 50 mM Tris HCl, pH 7.5, and .02% Brij-35. The gels were then stained in a 0.5% Coomassie Blue R250 solution for 3 hours and then destained in a solution containing 30% methanol and 10% acetic acid in water. The area of enzymatic degradation in each band was quantitated using a scanner interfaced to a Macintosh G3 personal computer. Band intensity was determined using the UN-SCAN-IT 4.3 program (Silk Scientific, Orem UT). Appropriate prestained size markers (Biorad, Hercules, CA) and laboratory standards allowed size estimation. To control for intergel variation, positive controls were run in duplicate on each gel and used to normal each band intensity.

Based on our previous data,21 10 women were required in each menopausal status group to detect a difference of at least 10% in the group-specific amounts of total collagen or the group-specific amounts of the predominant subtype (collagen III) for a power of 80% at the 0.05 significance level, using a 1-way analysis of variance. Because collagen levels were not normally distributed in our previous studies, the final sample size was increased by 10% to compensate for the use of nonparametric methods for analyzing the difference in median collagen levels between menopausal status groups. The primary outcome of the study was to detect a difference in the amount of total collagen or the amount of the predominant subtype (collagen III) in patients with and without prolapse. Secondary outcome measures included a change in collagen I or collagen V expression, an increase in MMP–2 and MMP–9 expression (pro and active forms), and a change in collagen fiber orientation in patients with prolapse and without prolapse. In addition, the associations between the primary and secondary outcome measures and menopausal status were evaluated. Finally, the associations between demographic variables listed under subject data collection with collagen or MMP expression were evaluated.

All statistical analyses were performed using SPSS 12.0.1 statistical software (SPSS Inc., Chicago, IL), and tests were evaluated at the 0.05 significance level. The levels of total collagen, collagen subtypes (I, III, and V), and MMPs in tissue did not seem to be normally distributed when graphically displayed. In addition, the values of the skewness and kurtosis statistics indicated a significant departure from symmetry. Therefore, nonparametric methods were used to evaluate the difference in medians. Differences in the median levels of total collagen, collagen subtypes (I, III, and V), and MMPs in the tissue between women with and without prolapse and between the 4 groups of women based on menopausal status were evaluated using the Mann-Whitney U and Kruskal-Wallis tests, respectively. When a significant overall association with menopausal status was detected, post-hoc pair-wise comparisons were made between the control group and the other 3 groups of women using the Mann-Whitney U test. The significance level at which the post-hoc tests were evaluated was adjusted to 0.017 using the Bonferroni method. The association of demographic variables with menopausal status and collagen and MMP levels were evaluated using Mann-Whitney U, Kruskal-Wallis, and analysis of variance where appropriate. Multivariate linear regression was used to identify factors that were independently associated with collagen and MMP levels.

RESULTS

The demographic characteristics of the patients recruited into the study are outlined in Table 1. The table is divided according to the protein that was analyzed in each subject's vaginal tissue – collagen or Matrix Metalloproteinases (MMPs). Among the women with prolapse, 11 patients had prolapse without incontinence while 26 patients had prolapse and incontinence concomitantly. Of the women who used hormones, 3 were on estrogen and progesterone and the remaining 10 women were on estrogen alone.

In both groups, the menopausal women were older; however, there was no difference in age between the premenopausal women with and without prolapse. In the collagen group there was no difference in gravidity, parity, BMI, and stage of prolapse among the 4 groups of women. In the MMP group, the premenopausal women with prolapse had a lower stage of prolapse than the menopausal women. The racial distribution in both groups was largely white, with 3 African-American women in the control groups for both collagen and MMPs and 1 African-American woman in the premenopausal prolapse group for collagen analysis.

Examination of trichrome-stained full-thickness vaginal biopsies using standard light microscopy techniques confirmed that all layers were present in each biopsy, with an epithelium, subepithelium, muscularis, and adventitia. Similar analysis after dissection of the epithelium and underlying peritoneum when present demonstrated that dissected sections consisted of the subepithelium, muscularis, and adventitia. In both groups, the amount of adventitia varied significantly, and therefore, our analysis focused primarily on the subepithelium and muscularis. Menopausal status and exogenous hormone use was evident from the appearance of the epithelium. An increase in cellular infiltrate was not apparent in prolapsed vaginal tissues relative to control tissues (Fig. 1). As shown in Figure 2, collagen bundles within the vaginal subepithelium were not parallel but instead seemed to be arranged in a whorled pattern. We were unable to detect any consistent qualitative differences in the orientation of collagen fibers in controls relative to cases or according to menopausal status.

Scanning confocal microscopy was used to analyze fluorescent micrographs simultaneously labeled with antibodies to collagen I, collagen III, and collagen V. By this method, we found that in subjects both with and without prolapse, collagen III is the predominant collagen throughout the vaginal wall (Fig. 3, Table 2). The amount of total collagen was significantly increased in patients with prolapse relative to controls. Post-hoc pair-wise comparisons between cases and controls demonstrated that the increase in collagen was most pronounced in premenopausal women (49% increase, P = .016). An increase in total collagen was also present in postmenopausal women not on hormone therapy (45% increase) but did not reach statistical significance when adjusting for multiple comparisons (P = .041). There was no difference in the amount of total collagen in postmenopausal women on hormone therapy and controls (P = .063).

Further analysis of collagen subtypes by immunofluorescence, demonstrated that the increase in total collagen in women with prolapse was primarily due to an increase in collagen III. Post hoc pair-wise comparisons between cases and controls showed that the increase in collagen III was most significant in premenopausal women (37% increase, P = .016) and postmenopausal women not on hormone therapy (45% increase, P = .015). In women on hormone therapy, there was no difference in the amount of collagen III relative to premenopausal women (P = .072). A comparison between premenopausal women with and without prolapse confirmed that both total collagen and collagen III were significantly higher in the women with prolapse relative to controls (Table 2; P = .016 and 0.016, respectively). This association was independent of age (data not shown, P > .05).

Because the most pronounced difference in collagen was between patients with prolapse and controls, we then combined the patients with prolapse into a single group independent of menopausal status. As shown in Table 3, by this analysis, the increase in collagen III between patients with and without prolapse was highly significant (P = .007). In addition, the increase in collagen V approached significance (P = .057).

As part of our secondary outcomes, we asked whether the increase in collagens was associated with a marker of increased tissue remodeling. To achieve this aim, we analyzed the expression of both the proenzyme and active forms of the fibrillar collagen degrading enzymes, matrix metalloproteinases–2 and –9 (MMP-2 and MMP-9) using quantitative substrate zymography. By this method we found that there was no difference in the expression of either the proenzyme or active forms of MMP-2 in patients with and without prolapse or according to menopausal status (Table 4). Similarly, the expression of proenzyme MMP-9 was not affected by prolapse or menopausal status. In contrast, the active form of MMP-9 was increased in patients with prolapse relative to controls (P= .030). Post-hoc pair-wise comparisons between cases and controls showed that the expression of active MMP-9 was significantly increased in premenopausal patients with prolapse (28% increase, P = .009) and in patients on standard doses of hormone therapy for at least 1 year (39% increase, P = .017). The increased expression in postmenopausal patients not on hormone therapy did not achieve significance after adjusting for multiple comparisons (P = .053). When patients with prolapse were combined into a single group independent of menopausal status and compared with controls, the increase in active MMP-9 expression was highly significant (Table 3, P = .005). The increase in active MMP-9 occurred independent of age (data not shown, P > .05).

Additional analyses were performed to determine whether any of the variables described under patient data collection were independently associated with collagen subtype or MMP expression. By this analysis, the expression of proMMP-2 increased with age when age was examined as a continuous variable (P = .01). The amount of total collagen was 23% lower in patients with prolapse and incontinence relative to patients with prolapse only (P = .038). Patients who were current smokers had 28% lower total collagen levels than past smokers and nonsmokers (P = .038). Patients with a parity of greater than 1 were found to have an increased amount of collagen I (P = .017). Finally, women who had a previous hysterectomy had increased expression of active MMP-9 (P = .040). Importantly, there was no association between stage of menstrual cycle and collagen or MMP expression.

DISCUSSION

To date, investigations into the changes in the vaginal wall that occur with prolapse are limited. Currently, the vast majority of studies comparing the vagina of women with and without prolapse have used biopsies of the vaginal wall or vaginal epithelium and a variable amount of subepithelial tissue that has been removed after repair of an anterior or posterior vaginal wall defect (ie, a colporrhaphy). Very few of the studies to date have confirmed the target tissue under study histologically, making it difficult to determine exactly which portion of the vaginal wall is being analyzed. Therefore, it is not surprising that the results have been inconsistent (8–15). The goal of the study outlined here was to define the changes in the vaginal wall that occur in women with prolapse using histologically defined specimens. We believe that this information will be useful to surgeons who perform reconstructive vaginal surgery. Our most important findings were that collagen III is the major fibrillar collagen of the vagina and the amount of collagen III and that the active form of MMP-9 is increased in the vagina of women with prolapse relative to that of controls.

Previously, it has been shown that collagen III is the predominant collagen subtype in the arcus tendineous fascia pelvis, a structure that plays an important role in providing support to the anterior vaginal wall21 and the uterosacral ligament, which provides apical support.22 Collagen III belongs to a family of fibril-forming collagens that are found throughout the body. Each of the fibrillar collagens has a unique structural profile that in turn imparts tissues with distinct physical properties.23,24 Within this family, collagen I forms large fibers and predominates in tissues with high tensile strength, including ligament, tendon, bone, and the rectus sheath. Collagen III, forming smaller fibers, predominates in tissues that require increased flexibility and distensibility and are subject to periodic stress such as the vasculature. Collagen V is a quantitatively minor collagen that forms small fibers. Although it has predominately been studied as the primary collagen that confers the physical properties to the cornea, it has also been found to be important in wound healing and directing fibrillogenesis.25,26

It is not surprising that the vaginal wall, which must accommodate sudden transient increases in intra-abdominal pressure and the passage of the fetus, is primarily comprised of collagen III. A high level of collagen I relative to collagen III would not be desirable, because it would provide the vagina with increased tensile strength similar to tendons and ligaments but at the expense of distensibility and flexibility. The role of collagen V as a minor collagen in the vaginal wall is not clear but may be to regulate fiber size and to maintain the physical properties of the vagina. It is possible that a predominance of collagen III may allow the vagina to distend for delivery and to be flexible in the context of increases in intra-abdominal pressure but also may make this tissue more vulnerable to injury and subsequent prolapse.

Previous studies have also demonstrated an increase in collagen III expression in patients with prolapse in other supportive tissues of the vagina. In a recent study, Gabriel et al22 compared biopsies of the uterosacral ligament in women with and without prolapse and found an increase in the expression of collagen III in the prolapse group. Ewies et al27 reported an increase in collagen III in the cardinal ligament of patients with prolapse. Similar to our study, the increase in collagen III in both studies was observed independent of age.22,27 Neither study examined MMP expression. Jackson et al10 examined the vaginal epithelium of 8 premenopausal women with prolapse (with and without urinary incontinence) relative to 10 control women and found a decrease in total collagen and an increase in MMP expression. It is difficult to compare the results of this study to ours because we definitively excluded the epithelium from our analysis. Although their biopsies were not defined histologically, if Jackson and colleagues truly focused on the epithelium, it may account for the differences in total collagen observed in the 2 studies. It is also important to note that Jackson measured hydroxyproline relative to tissue dry weight as an indicator of total collagen in a sample, whereas we used an antibody-directed method and normalized to tissue area. Our goal was to perform a highly quantitative assay that would allow us to focus in on a specific area within the vaginal wall.

Matrix metalloproteinases are the key regulators of connective tissue degradation and therefore are involved in myriad physiologic and pathologic processes, including tissue remodeling, morphogenesis, wound healing, tumor invasion, and tumor metastasis.28–30 In addition, MMPs regulate many biologic processes through the release, activation, or sequestration of growth factors, growth factor binding proteins, cell surface receptors, and cell-cell adhesion molecules (reviewed by Mott et al30). Although MMP-2 is a ubiquitous, largely constitutively expressed enzyme, the expression of MMP-9 tends to be more localized and regulated. The activity of MMPs is regulated by endogenous inhibitors referred to as the tissue inhibitors of MMPs or tissue inhibitors of metalloproteinase (TIMPs), as well as the process of autocatalysis.31 The fibrillar collagens are initially degraded into three-quarter and one-quarter fragments by MMPs-1, -8, and –13 before their further breakdown into soluble fibers by MMPs-2 and -9. In a previous study of vaginal wall biopsies, the expression of MMP-1 mRNA was found to be increased in patients with prolapse and incontinence, whereas the expression of TIMP-1 mRNA was shown to be decreased.14 Total collagen protein measured as hydroxyproline per milligram of protein was also decreased. Although the biopsies in this study were not characterized histologically, they were measured to range in thickness from 5–8 mm. Thus, it is likely that they all included epithelium but varying amounts of subepithelium and muscularis, obviating a direct comparison to our study. Although we did not investigate the expression of inhibitors of MMPs, we acknowledge the potentially important role of TIMPs in the regulation of tissue remodeling in the vaginal wall. Tissue inhibitors of metalloproteinase will certainly be a focus of our future studies.

Our finding of an increase in the expression of both collagen and matrix metalloproteinase expression in prolapsed tissue is typical of a tissue that is remodeling after injury32 or a tissue that is remodeling to accommodate a progressively increasing mechanical load.33 After injury to the levator ani muscles or disruption of connective tissue attachments at child birth, transient increases in intra-abdominal pressure may result in a greater proportion of the load being transmitted to the vaginal wall. Indeed, connective tissue is known to be a highly dynamic structure that adapts in both a structural and functional way to increased mechanical loading. Increased mechanical loads have been shown to accelerate connective tissue remodeling in soft tissues throughout the body. The effect of remodeling varies according to the target system, with detrimental effects observed in the heart (eg, cardiomyopathy34,35) and beneficial effects seen in the musculoskeletal system (improved loading in response to exercise36,37). An increase in MMP-9 specifically has been associated with tissue remodeling in bone,38 coronary artery,39,40 and healing dermal wounds.41

It is likely that after injury to its supportive structures, the vaginal wall remodels to accommodate the biomechanical pressures of the prolapsing organs. Thus, the increase in collagen III and MMP expression more likely reflects the secondary effects of prolapse on the tissue rather than a host predisposition to prolapse. In either case, it is expected that the increase in collagen III and collagen breakdown negatively affects the biomechanical properties of the prolapsed vagina, which in turn may render the native tissue suboptimal for prolapse repairs. Future studies aimed at comparing the mechanical properties of prolapsed and nonprolapsed vaginal tissue will clarify this issue.

We did not find a correlation between increased collagen or MMP expression and increased stage of prolapse. Indeed, one would predict that women with more severe prolapse (stage IV) would have the highest expression of these proteins, because the biomechanical stresses on their tissues are the highest. We performed our analysis, however, according to the maximal stage of prolapse. It is very likely that stage of prolapse is not a sensitive enough measurement to detect the effect of severity of prolapse on the vaginal wall. This is especially true in distinguishing between patients whose prolapse differs by 1 stage. One could argue that a control patient with a stage I prolapse 1.5 cm above the hymen is not that different from a prolapse patient with a stage II prolapse 1.0 cm above the hymen. It is likely that specific points measured in centimeters during a Pelvic Organ Prolapse Quantification examination will prove to be a more sensitive measure. In this study, some of the patients were examined using the Baden Walker classification, precluding such an analysis. It is also important to point out that the vast majority of women in the study had stage II or III prolapse (n = 54), with very few women having stage IV (n = 4). A larger sample size that includes more women with stage IV prolapse may also help to resolve this issue.

An additional limitation of this study is the absence of an appropriate control group for menopausal women with prolapse. At our institution, the vast majority of postmenopausal women who undergo pelvic surgery for a benign indication have prolapse; otherwise, it is likely that a postmenopausal woman is having pelvic surgery for a premalignant condition (endometrial hyperplasia) or a malignancy. We required that our postmenopausal patients have no bleeding for more than 1 year, which further excludes the small number of patients who may have a hysterectomy for postmenopausal bleeding. At this point, we do not have permission to obtain a full-thickness vaginal biopsy from patients undergoing a laparoscopy (benign adnexal mass). Therefore, we were unable to obtain a postmenopausal control group. Others have published on this problem as well.15 To take into account the effect of age, however, we examined age as an independent variable in a multivariate regression model to determine whether it had an effect on any of the outcomes achieving significance. By this method, neither the increase in collagen III nor the increase in active MMP-9 was affected by age.

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